Craniofacial skeletal development is specified by a coordinated temporal and spatial pattern of gene expression. It involves both intramembranous and endochondral ossification. Ultimately, secreted morphogens such as transforming growth factor beta, bone morphogenetic proteins, hedgehog proteins and wingless proteins signal to key transcription factors to specify gene expression. The comparative phenotypic study of human and mouse genetic craniofacial skeletal malformation syndromes is a powerful approach for dissecting these signaling pathways. Two such syndromes, cleidocranial dysplasias (CCD) and trichorhinophalangeal syndrome (TRPS) are caused by mutations in transcription factors RUNX2 and TRPS1 that are important in craniofacial skeletogenesis. The two phenotypes exhibit distinct as well as common clinical features that point to potential biochemical and genetic interaction. Based on our preliminary studies, we hypothesize that TRPS1 serves a critical function in specifying chondrogenic mesenchymal condensations and chondrocyte differentiation including during chrondrocyte hypertrophy, though additional functions in proliferating chondrocytes and/or bone cannot yet be excluded. Furthermore, we hypothesize that Trpsl may accomplish this by modifying RUNX2 function in an antagonistic fashion. Our Specific Aims are to 1) characterize the craniofacial skeletal and dental phenotype of already generation Trpsl mutant mice on a histological and molecular level, 2) evaluate the consequence of overexpression of Trps1 in proliferating and hypertropic chondrocytes in vivo, 3) determine the context dependence of TRPS1 repression of RUNX2 both in vivo with transgenic mice and in vitro with cell models of osteoblast differentiation, and 4) determine whether TRPS1 and RUNX2 interact genetically by generating double mutant animals and biochemically by immunoprecipitation and protein binding studies. These studies will answer two important questions: What is the function of TRPS1 during craniofacial skeletal development? How does TRPS1 regulate RUNX2 activity? By combining human clinical genetics with mouse genetic and cellular approaches, these studies will provide new perspectives into the transcriptional mechanisms that govern craniofacial skeletal development including mesenchymal cell fate commitment and regulation of chrondrocyte hypertrophy.